The present invention relates to the production of hybrid paucilamellar lipid vesicles. More particularly, the present invention concerns lipid vesicles which have phospholipids or glycolipids in addition to single-chain non-ionic, anionic, or zwitterionic surfactants as the major components of the walls (or lipid bilayers) of a paucilamellar lipid vesicle.
Lipid vesicles are substantially spherical structures made of materials having a high lipid content, e.g., surfactants or phospholipids. The lipids of these spherical vesicles are organized in the form of lipid bilayers. The lipid bilayers encapsulate an aqueous volume which is either interspersed between multiple onion-like shells of lipid bilayers (forming multilamellar lipid vesicles or "MLV") or the aqueous volume is contained within an amorphous central cavity. The most commonly known lipid vesicles having an amorphous central cavity filled with aqueous medium are the unilamellar lipid vesicles. Large unilamellar vesicles ("LUV") generally have a diameter greater than about 1 .mu. while small unilamellar lipid vesicles ("SUV") generally have a diameter of less than 0.2 .mu.. Lipid vesicles have a variety of uses including adjuvants or carriers for a broad spectrum of materials.
Although substantially all the investigation of lipid vesicles in recent years has centered on multilamellar and the two types of unilamellar lipid vesicles, a fourth type of lipid vesicle, the pauoilamellar lipid vesicle ("PLV"), exists. See Callo and McGrath, Cryobiology 1985, 22(3), pp. 251-267. This lipid vesicle has barely been studied until recently and had only been manufactured with phospholipids surrounding an amorphous aqueous-filled volume. PLV's consist of about 2 to 10 peripheral bilayers surrounding a large, unstructured central cavity. In all PLV's described previous to U.S. patent application Ser. No. 157,571, now U.S. Pat. No. 4,911,928 the disclosure of which is incorporated herein by reference, this central cavity was filled with an aqueous solution. The cited application first disclosed oil-filled vesicles.
Each type of lipid vesicle appears to have certain uses for which it is best adapted. For example, MLV's have a higher lipid content then any of the other lipid vesicles, so to the extent that a lipid vesicle can encapsulate or carry a lipophilic material in the bilayers without degradation, MLV's have been deemed the most advantageous for carrying lipophilic materials. In contrast, the amount of water encapsulated in the aqueous shells between the lipid bilayers of the MLV's is much smaller than the water which can be encapsulated in the central cavity of LUV's, so LUV's have been considered advantageous in transport of aqueous material. However, LUV's, because of their single lipid bilayer structure, are not as physically durable as MLV's and are more subject to enzymatic degradation. SUV's have neither the lipid or aqueous volumes of the MLV's or LUV's but because of their small size have easiest access to cells in tissues.
PLV's, which can be considered a sub-class of the MLV's, possess features of both MLV's and LUV's. PLV's appear to have advantages as transport vehicles for many uses as compared with the other types of lipid vesicles. In particular, because of the large unstructured central cavity, PLV's are easily adaptable for transport of large quantities of aqueous- or oil-based materials. Moreover, the multiple lipid bilayers of the PLV's provides PLV's with additional physical strength and resistance to degradation as compared with the single lipid bilayer of the LUV's. As illustrated in the present application and the previously cited U.S. patent application Ser. No. 157,571, now U.S. Pat. No. 4,911,928 the central cavity of the PLV's can be filled wholly or in part with an apolar oil or wax and then can be used as a vehicle for the transport or storage of hydrophobic materials. The amount of hydrophobic material which can be transported by the PLV's with an apolar core is much greater than can be transported by MLV's.
Conventional methods for producing multilamellar lipid vesicle start by dissolving the lipids, together with any lipophilic additives, in an organic solvent. The organic solvent is then removed by evaporation using heat or by passing a stream of an inert gas (e.g., nitrogen) over the dissolved lipids. The residue is then hydrated with an aqueous phase, generally containing electrolytes and additives such as hydrophilic biologically-active materials, to form multilamellar lipid membrane structures. In some variations, different types of particulate matter or structures have been used during the evaporation process to assist in the formation of the lipid residue. Changing the physical structure of the lipid residue can result in formation of better vesicles upon hydration. Two recent review publications, Gregoriadis, G., ed. Liposome Technology (CRC, Boca Raton, Fla.), Vols. 1-3 (1984), and Dousset and Douste-Blazy (in Les Liposomes, Puisieux and Delattre, Editors, Techniques et Documentation Lavoisier, Paris, pp.41-73 (1985)), summarize the methods which have been used to make MLV's.
No matter how the MLV's or PLV's are formed, once made it is necessary to determine the effectiveness of the process. Two measurements commonly used to determine the effectiveness of encapsulation of materials in lipid vesicles are the encapsulated mass and captured volume. The encapsulated mass is the mass of the substance encapsulated per unit mass of the lipid and is often given as a percentage. The captured volume is defined as the amount of the aqueous phase trapped inside the vesicle divided by the amount of lipid in the vesicle structure, normally given in ml liquid/g lipid.
Phospholipid vesicles, while mimicking membrane structure because of similarity of materials with naturally occurring membranes, have a number of problems. First, isolated phospholipids are subject to degradation by a large variety of enzymes. Second, the most easily available phospholipids are those from natural sources, e.g., egg yolk lecithin, which contain polyunsaturated acyl chains that are subject to autocatalyzed peroxidation. When peroxidation occurs, the lipid structure breaks down, causing fracture of the lipid vesicle and premature release of any encapsulated material. While hydrogenation may be used to saturate the chains, it is an expensive process which raises the already high cost of the phospholipid starting materials, as well as changing the vesicle stability.
Because of these problems with using plain phospholipids, certain companies, primarily L'Oreal and Micro Vesicular Systems, have been using non-ionic surfactants to form the structure of vesicles. L'Oreal uses primarily polyglycols, e.g, U.S. patent Ser. Nos. 4,772,471 and 4,217,344, while Micro Vesicular Systems has been using primarily polyoxyethylene fatty acid ethers and esters (see U.S. patent application Ser. No. 157,571 now U.S. Pat. No. 4,911,928 and U.S. Pat. No. 4,855,090). The L'Oreal vesicles appear to be classic MLV's while the Micro Vesicular Systems vesicles are primarily PLV's.
For certain uses, e.g., transportation of vesicles through membranes or permeation of the skin, the presence of a small amount of phospholipid and/or glycolipid to the bilayer structure of the vesicles may be important. A problem with using the phospholipids or glycolipids in conjunction with many synthetic surfactants is that most of the surfactants have a non-ionic head group linked to a single hydrophobic chain while most phospholipids and glycolipids have two hydrophobic chains linked to an ionic head group. Use of both single and multiple chain molecules in the structure of vesicle walls may lead to problems in the packing of the lipids which form the lipid bilayers. Under most circumstances, one would expect that any attempt to form a stable vesicle by blending single and multiple chain lipids, particularly when one is a non-ionic lipid while the other is an ionic or zwitterionic lipid, would be difficult at best. In addition, the phospholipids are still subject to phospholipases after vesicle formation. However, the hybrid vesicles are exactly what is needed to solve certain problems of cross-membrane transport, stability and cost.
Accordingly, an object of the invention is to provide stable hybrid lipid vesicles having a non-ionic, zwitterionic, or anionic surfactant and a phospholipid or glycolipid in the lipid bilayers of the vesicles.
A further object of the invention is to provide stable hybrid paucilamellar lipid vesicles encapsulating a water-immiscible material within the central amorphous cavities of the vesicles.
Another object of the invention is to provide a method of manufacture of hybrid vesicles.
A still further object of the invention is to provide a vehicle for the transport of oil-soluble or water-soluble materials into the skin.
These and other objects and features of the invention will be apparent from the following description.